Project Summary Maintenance of proper patterns of DNA methylation is essential for the integrity of cell identity, failure of which results in aberrant activation of genes, which in turn modulates signaling pathways leading to cancer and de- velopmental disorders. Despite a large body of evidence supporting the role of aberrant DNA methylation in etiology of several human diseases, the fundamental mechanisms that regulate the target site specificity of the de novo DNA MTases, Dnmt3a and 3b, are largely unknown. Gene repression is an orchestrated event that involves loss of coactivator complexes from the regulatory elements and changes in chromatin state including gain of DNA methylation, resulting in stable gene repression. Recent studies have enumerated the role of en- hancer-mediated regulation of oncogenes in various cancers. Others have shown aberrant expression of plu- ripotency genes mediates dedifferentiation in several cancers. Changes in the chromatin state of the enhanc- ers of pluripotency genes have been shown to be critical for the repression of pluripotency genes during mu- rine embryonic stem cell (ESC) differentiation. The role of DNA methylation in this process and the mechanism that targets DNA methylation to the enhancers during differentiation have not been addressed. Understanding the fundamental epigenetic mechanisms involved in the establishment of enhancer-mediated pluripotency (Pp) gene repression during normal cell differentiation will, in the long-term, lead to development of therapeutic strategies to restore Pp gene repression in cancer cells. Our objective in this application is to elucidate molecu- lar mechanism(s) that regulate the activity of Dnmt3a and 3b at enhancers of Pp genes genome-wide using ESC differentiation as a model system. Supported by our strong preliminary data and recently published stud- ies on the dynamics of the chromatin state of Pp gene enhancers, we will test our hypothesis that Lsd1- Mi2/NURD activity acts as an epigenetic switch at Pp gene enhancers to activate Dnmt3 enzymes, causing site-specific DNA methylation and stable Pp gene repression. We will further elucidate the role of chromatin conformation in facilitating enhancer-targeted activity of Dnmt3a and/or 3b to specific promoters during the ear- ly phase of ESC differentiation. To test if both Dnmt3a and 3b are regulated by these mechanisms, we will map their genome-wide activity during ESC differentiation. Our rationale for these studies is that their successful completion is expected to fill the gap in our understanding of how the epigenetic ?cross talk? mechanisms, dur- ing cell differentiation, modulate Dnmt3a/3b activity to terminate the pluripotency program, disruption of which could lead to developmental disorders and cancer. Additionally, the outcomes from these studies are expected to provide new insights into how the interplay between various epigenetic factors affects gene expression, con- tributing to phenotypic variation and transgenerational inheritance.